regulatory t cells in ovarian cancer are characterized by ......biology of human tumors regulatory t...

Biology of Human Tumors

Regulatory T Cells in Ovarian Cancer AreCharacterized by a Highly Activated PhenotypeDistinct from that in MelanomaAras Toker1, Linh T. Nguyen1, Simone C. Stone1, S.Y. Cindy Yang2, Sarah Rachel Katz3,Patricia A. Shaw4, Blaise A. Clarke4, Danny Ghazarian4, Ayman Al-Habeeb4,Alexandra Easson5,Wey L. Leong5, David R. McCready5, Michael Reedijk1,2,5,Cynthia J. Guidos6,7, Trevor J. Pugh2,8, Marcus Q. Bernardini3, and Pamela S. Ohashi1,2,6

Abstract

Purpose: Regulatory T (Treg) cells expressing the transcrip-tion factor FOXP3 are essential for the maintenance of immu-nologic self-tolerance but play a detrimental role in mostcancers due to their ability to suppress antitumor immunity.The phenotype of human circulating Treg cells has beenextensively studied, but less is known about tumor-infiltratingTreg cells. We studied the phenotype and function of tumor-infiltrating Treg cells in ovarian cancer and melanoma toidentify potential Treg cell–associated molecules that can betargeted by tumor immunotherapies.

Experimental Design: The phenotype of intratumoraland circulating Treg cells was analyzed by multicolorflow cytometry, mass cytometry, RNA-seq, and functionalassays.

Results: Treg cells isolated from ovarian tumors displayeda distinct cell surface phenotype with increased expression of a

number of receptors associated with TCR engagement, includ-ingPD-1, 4-1BB, and ICOS.Higher PD-1 and4-1BB expressionwas associated with increased responsiveness to further TCRstimulation and increased suppressive capacity, respectively.Transcriptomic and mass cytometry analyses revealed thepresence of Treg cell subpopulations and further supporteda highly activated state specifically in ovarian tumors. Incomparison, Treg cells infiltrating melanomas displayedlower FOXP3, PD-1, 4-1BB, and ICOS expression and wereless potent suppressors of CD8 T-cell proliferation.

Conclusions: The highly activated phenotype of ovariantumor-infiltrating Treg cells may be a key component of animmunosuppressive tumor microenvironment. Receptorsthat are expressed by tumor-infiltrating Treg cells could beexploited for the design of novel combination tumor immu-notherapies. Clin Cancer Res; 1–12. �2018 AACR.

IntroductionThe unprecedented success of immune checkpoint inhibitors

has fueled efforts to further explore, expand, and develop novelcancer immunotherapy approaches. However, the mechanismof action of checkpoint inhibitors is not clear and responserates are highly variable, and typically lower than 50%, acrossdifferent cancers (1, 2). Therefore, research has been intensified

to uncover the mechanism of action and to identify biomarkersassociated with response or resistance in order to establishguidelines for patient selection and provide a rationale for thedevelopment of combination immunotherapies (3, 4).Mechanisms of immune suppression within the tumor micro-environment have been of particular focus, including the upre-gulation of inhibitory molecules and the recruitment of sup-pressive cell populations such as myeloid-derived suppressorcells (MDSC), tumor-associated macrophages (TAM), and reg-ulatory T (Treg) cells (5–7).

Treg cells are essential for the maintenance of immunologicself-tolerance and the prevention of autoimmunity (8). Thetranscription factor FoxP3 is a master regulator of Treg cellfunction, and its deficiency leads to devastating autoimmunepathology in mice (8) and humans (9). In mice expression ofFoxP3 protein is strictly restricted to bona fide suppressive Tregcells, whereas in humans, effector T cells without suppressivefunction can also transiently express low levels of FoxP3 uponactivation (10). Among many mechanisms Treg cells deploy toexhibit their immunosuppressive function are direct lysis ofimmune effector cells, depletion of growth factors and metabo-lites, inhibition of antigen-presenting cell function, and secretionof regulatory cytokines (9).

The majority of circulating Treg cells belong to a distinctand phenotypically stable lineage of CD4þ T cells in mice andhumans, as the expression of the master transcription factorFoxP3 is initiated and epigenetically imprinted during thymic

1The Campbell Family Institute for Breast Cancer Research, Princess MargaretCancer Centre, University Health Network, Toronto, Ontario, Canada.2Department of Medical Biophysics, University of Toronto, Toronto, Ontario,Canada. 3Division of Gynecologic Oncology, University Health Network,Toronto, Ontario, Canada. 4Department of Laboratory Medicine and Pathobi-ology, University Health Network, University of Toronto, Toronto, Ontario,Canada. 5Department of Surgical Oncology, University HealthNetwork, Toronto,Ontario, Canada. 6Department of Immunology, University of Toronto, Toronto,Ontario, Canada. 7Program inDevelopmental and StemCell Biology, Hospital forSick Children Research Institute, Toronto, Ontario, Canada. 8Princess MargaretGenomics Centre, University Health Network, Toronto, Ontario, Canada.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

CorrespondingAuthor: Pamela S. Ohashi, Princess Margaret Cancer Centre, 610University Avenue, Suite 8-407, Toronto, Ontario, Canada M5G 2C9. Phone: 416-946-4501, ext. 3689; Fax: 416-204-2276; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-0554

�2018 American Association for Cancer Research.

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development (11, 12). However, a subset of Treg cells alsoretains plasticity and can revert to a phenotype with diminishedsuppressive function and produce IFNg or IL17 under inflam-matory conditions (13, 14). FOXP3þ-suppressive Treg cells canalso be induced in vivo from FOXP3� conventional T cells bystimulation in the presence of TGFb (15). Hence, the pool ofcirculating Treg cells consists of thymus-derived and peripherallyinduced subsets, and the relative contribution of each subset tostable and plastic Treg cell subpopulations has not been exten-sively studied.

Treg cells accumulate at the tumor site in many cancersincluding head and neck, colorectal, liver, lung, breast, ovarian,skin, and pancreas (5). A high prevalence of Treg cells withintumor-infiltrating lymphocytes (TIL) is associated with a pooroutcome, in particular when combined with poor infiltrationby CD8þ cytotoxic T cells, in most cancers including ovarian(5, 16). In line with this, experimental depletion of Treg cellsfosters immune responses against established tumors in murinemodels (17, 18). Therefore, the idea of interfering with Tregcell function has gained much interest for the treatment ofhuman cancers.

The cell surface expression of various immunoglobulin andTNFR superfamily members by human circulating Treg cells hasbeen well defined (19), but potential changes to the surfacephenotype following recruitment to tumorshas not been carefullycharacterized across cancers. Because many receptors such asPD-1, 4-1BB, and ICOS represent potential targets for immuno-therapy, we investigated their expression by intratumoral Tregcells from patients with ovarian cancer andmelanoma. We foundthat Treg cells infiltrating ovarian tumors have a highly activatedphenotype with potent suppressive activity and enhanced expres-sion of both costimulatory and inhibitory receptors, which couldpotentially be exploited for targeted Treg cell modulation. Incontrast, the phenotype of melanoma-infiltrating Treg cells wasindicative of a lower activation state, which could contribute tothe higher response rates to immunotherapy in melanoma.

Materials and MethodsStudy participants

All human tissue and blood were obtained through protocolsapproved by the Institutional Review Board in concordance withthe Declaration of Helsinki. Written informed consent wasobtained from all donors who provided the samples.

Tumor specimensTumor specimens were obtained from the UHN Biospecimen

Program, through surgical resection from patients diagnosedwith epithelial ovarian cancer (mostly high-grade serous) ormelanoma. Patient characteristics are summarized in Supplemen-tary Table S1. Fresh tumor specimenswere enzymatically digested(RPMI1640 containing 2 mmol/L L-glutamine, 1 mg/mL colla-genase, 10 mg/mL Pulmozyme, 100 U/mL penicillin, 100 mg/mLstreptomycin, 10 mg/mL gentamicin sulfate, and 1.25 mg/mLAmphotericin B) using theGentleMACS system (Miltenyi Biotec).Tumor digests were cryopreserved in human serum containing10% DMSO prior to analysis. A total of 2.2 � 107 (�1.7 � 107)and 1.1� 107 (�1.3� 107) cells were processed and analyzed perovarian tumor and melanoma specimen, respectively. Cell via-bility for the ovarian and melanoma specimens was 61.7%(�22.4%) and 43.0% (�29.1%), respectively (SupplementaryTable S1).

Flow cytometry and cell sortingCells were stained with anti-CD3 (clone OKT3), anti-CD4

(RPA-T4), anti-CD8 (RPA-T8), anti–PD-1 (eBioJ105), anti–4-1BB(4B4-1), anti–TIM-3 (344823), anti-OX40 (Ber-ACT35), anti-TIGIT (MBSA43), anti–DNAM-1 (DX11), anti-CD27 (O323),anti-CD28 (CD28.2), anti-CD25 (M-A251), anti-ICOS (C398.4A),anti-CD45RA (HI100), and anti-CD127 (A019D5). Cells werefixed and permeabilized with the Foxp3 transcription factorstaining buffer set followed by intracellular staining with anti-FOXP3 (236A/E7) and anti–CTLA-4 (BNI3). Samples wereacquired on a BD LSR Fortessa SORP flow cytometer and datawere analyzed using the FlowJo software.

Cell sortingwas performed on a BDFACSAriaII or BDFACSAriaFusion SORP cell sorter by The Flow andMass Cytometry Facility,The Hospital for Sick Children, Toronto, Canada. IntracellularFOXP3 staining was performed on an aliquot of sorted cells asabove.

Mass cytometryPurified mAbs were conjugated with heavy metals by the Flow

and Mass Cytometry Facility, The Hospital for Sick Children.Whole tumor digests were counted and 2 � 106 cells for eachsample were stained for cell surface markers (SupplementaryTable S3) in staining media (PBS containing 1% BSA and0.02% NaN3) for 30 minutes at 4�C. Cells were washed withprotein-free PBS, stained with 1 mmol/L cisplatin for 5 minutes atroom temperature, fixed using the transcription factor buffer set(BDBiosciences) followed by intracellular staining for 60minutesat 4�C. Cells were washed with staining media and stained with100 nmol/L iridium-labeled DNA-intercalator (Fluidigm) in PBScontaining 0.3% saponin and 1.6% formaldehyde at 4�C for up to48 hours. Cells were washed twice with deionized water prior toadding EQ normalization beads containing Ce140, Eu151,Eu153, Ho165, and Lu175 (Fluidigm) and acquiring on a Heliosmass cytometer by The Flow and Mass Cytometry Facility, The

Translational Relevance

Rates of response to immunotherapy in ovarian cancer arelow despite pronounced immune infiltration. RegulatoryT (Treg) cells infiltrate many human tumors and are in mostcases associated with poor prognosis. In this study, wefound that tumor-infiltrating Treg cells express a distinctarray of targetable immunoreceptors, and are highly acti-vated and potently suppressive. Melanoma-infiltrating Tregcells share many features of this Treg cell phenotype, butpossess distinct differences that could require reconsidera-tion and adjustment of immunotherapeutic approachesaccording to cancer type. A highly suppressive tumor micro-environment could also explain the low response rate inovarian cancer and suggests that combination therapies thatboost antitumor immunity and diminish immunosuppres-sion could be more promising. The Treg cell expressionpattern of inhibitory receptors could become an importantfactor to consider in treatment decisions to avoid unwantedincreases in Treg cell–mediated immunosuppression bycytotoxic T-cell–targeted immunotherapy regimens.

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Hospital for Sick Children. After normalizing and randomizingvalues near zero using the Helios software, FCS files wereuploaded to Cytobank for analysis. Data were initially visualizedon bivariate plots with hyperbolic arcsine axes using scale argu-ments between 2 and 5. Pregating was performed as illustrated inSupplementary Fig. S4A. ViSNE and SPADE analyses were per-formed on pregated CD3þ or CD3þCD4þCD8� cells using 23 to24 mapping parameters (Supplementary Table S3). ViSNE anal-ysis was performed with equal sampling (6,000–20,000 CD3þ

events from each sample), and perplexity was set to 30 and thetato 0.5. Final KL divergence values of 2.60 to 3.37 were achievedafter a total of 1,000 iterations. The SPADE algorithm was runusing the same mapping parameters (Supplementary Table S3)without downsampling and target nodes were set to 30 to 50.

Suppression assayPeripheral bloodmononuclear cells (PBMC)were labeled with

the eFluor450 Cell proliferation dye and CD8þ responder cellswere purified by FACS. Suppressor cells (Treg or control) fromtumor specimens were FACS-purified. PBMCs were depleted ofT cells using a PE-conjugated anti-CD3 (clone UCHT1) antibodyand the EasySep Human PE Positive Selection Kit (StemcellTechnologies). Responder cells and Treg cells were combined toa total of 2.5 � 104 T cells at the indicated ratio of CD8þ T cellsand Treg or control cells and activated with 5 mg/mL immobilizedanti-CD3 (clone OKT3) in the presence of 7.5 � 104 T cell–depleted PBMCs. Cells were harvested 4 days later and dilutionof the proliferation dye on live CD8þ cells was analyzed byflow cytometry. Suppression was calculated using the followingformula: % suppression ¼ 1 � [division index (responder þsuppressor cells)/division index (responder cells alone)] � 100.

Treg cell activation assayTreg cellswere FACS-purified as above and cultured at 2.0�104

cells per well in a 96-well round-bottom plate. Tosyl-activatedDynabeads (Thermo Fisher Scientific) were coated with anti-CD3(clone OKT3) and anti-CD28 (CD28.2) either at a high concen-tration (10 mg each per 108 beads) or at a low concentration(1 mg each) according to themanufacturer's instructions, and Tregcells were stimulated by the addition of 2 � 104 Dynabeads and10 IU/mL rhIL2. One day after activation, cell surface expressionof 4-1BB and CD3 was analyzed by flow cytometry.

RNA-seqRNA libraries were prepared using SMARTer Stranded Total

RNA-seqKit-Pico InputMammalian (Clontech Laboratories). Thepaired-end libraries were sequenced on NextSeq 500 (Illumina)for 75 cycles. RNA sequencing (RNA-seq) was performed by thePrincess Margaret Genomics Centre (Toronto, Canada). Gene-level transcript expression and read counts were quantified usingRSEM (v1.2.29) with Gencode transept annotation (v26). Differ-entially expressed genes were identified using DESeq2 withdefault parameters to normalize and compare between gene-wiseread counts of defined sample groups. Genes with a minimum of2-fold expression difference and FDR-corrected P-value <0.1 arereported as statistically significant. Log-transformed gene expres-sion values [log2(TPMþ1)] were used to generate heat maps ofselected signature genes or differentially expressed genes. Princi-pal component analysis (PCA) was performed using library-sizenormalized gene-wise read counts from the top 500 genes withgreatest variance.

DNA methylation analysisBisulfite conversion of genomic DNA was performed with

the EZDNAMethylation Kit (Zymo). Converted DNAwas ampli-fied with the primers 50-TGATTTGTTTGGGGGTAGAGGATT-30

and 50-ACACCCATATCACCCCACCTAA-30, cloned into thepCR4-TOPO vector (Invitrogen) and sequenced by the SickKidsTCAG DNA sequencing facility.

Statistical analysisStatistical significance was determined by two-tailed unpaired

Mann–Whitney U test or unpaired t test. The n values used tocalculate statistics are defined and indicated in figure legends.Significance is indicated within figures, and differences that werenot significant (P > 0.05) are denoted ns. Multiple hypothesistesting was performed using Sidak multiple comparisons test.

ResultsOvarian tumors are infiltratedby suppressive FOXP3þTreg cellsthat express a distinct array of immunoreceptors

Tumor-infiltrating Treg cells are potent immune suppressors,and a high frequency of intratumoral Treg cells is associatedwith apoor outcome in epithelial ovarian cancers (16). We thereforeaimed to expand the knowledge on intratumoral Treg cells byanalyzing their expression of costimulatory and inhibitory recep-tors in ovarian cancer. We initially observed that intratumoralCD3þCD4þFOXP3þCTLA-4hi cells expressed PD-1 (Fig. 1A). Wefurther observed that intermediate levels of PD-1 expressiondefined a distinct subset of FOXP3-expressing CD4þ T cells(Fig. 1B). Importantly, circulating FOXP3þ cells from patientswith ovarian cancer did not express PD-1 (Fig. 1C). ICOSwas alsoupregulated by tumor-infiltrating FOXP3þ cells (Fig. 1D and E).Therefore, Treg cells from ovarian cancer have upregulatedTCR-induced genes, including PD-1 and ICOS.

To assess whether PD-1intFOXP3þ cells had regulatory func-tion, we evaluated strategies to purify this population. The com-monly used combination of CD25 and CD127 (to isolateCD25þCD127lo cells) clearly had its limitations owing to theheterogeneity of CD25 expression by intratumoral FOXP3þ cells(Supplementary Fig. S1A and S1B). Using PD-1 and ICOSwe could distinguish three main subsets of CD3þCD4þ TIL:PD-1hiICOSint, PD-1intICOShi and PD-1�ICOS�. Among thesesubsets FOXP3-expressing cells were contained within thePD-1intICOShi population (Supplementary Fig. S1C). In addition,the PD-1intICOShi population overlapped with FOXP3þ cells to asimilar extent as CD25þCD127lo (Fig. 1F–H). Importantly, gatingon PD1intICOShi did not introduce bias in favor of Treg cellsubpopulations expressing CD25 or 4-1BB, whereas theCD25þCD127lo gate was enriched for cells that expressed CD25and 4-1BB (Supplementary Fig. S1D and S1E). Therefore,the use of CD25 for cell sorting could bias the analysis ofintratumoral FOXP3þ cells and this could be overcome by usingPD-1 and ICOS.

Because human effector T cells can upregulate FOXP3following TCR engagement (10), we sought to confirm theTreg cell identity of intratumoral FOXP3þ cells directly throughfunctional assays as well as epigenetic and transcriptomicanalyses. Tumor-derived PD-1intICOShi cells could suppressproliferation of CD8þ T cells in a dose-dependent manner,whereas PD-1hiICOSint cells were devoid of suppressive capac-ity (Fig. 1I). FOXP3þ Treg cells show DNA demethylation at the

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Figure 1.

Tumor-infiltrating Treg cells in ovarian cancer express PD-1 and ICOS. A, Expression of PD-1 by CD3þCD4þFOXP3þCTLA-4hi TILs. B and C, Expression ofPD-1 on FOXP3þ TILs was analyzed by flow cytometry. Representative staining on gated CD3þCD4þ TILs (B) and data summarizing PD-1 expression levelson FOXP3þ cells from PBMCs and TILs (C) are shown (n¼ 10).D and E, Expression of ICOS on FOXP3þ TILswas analyzed by flow cytometry. Representative stainingon gated CD3þCD4þ TILs (D) and data summarizing percentage of ICOS-expressing cells within the FOXP3þ gate in PBMCs and TILs (E) are shown (n ¼ 10).Error bars, SD. �� , P < 0.0001, Mann–Whitney U test. F, Gating strategies for purifying intratumoral FOXP3-expressing cells using PD-1 and ICOS (top) orCD25 and CD127 (bottom). Plots are gated on CD3þCD4þ. G, Percentage of FOXP3þ cells within the Treg cell gates as in F. H, ViSNE analysis on flow cytometrydata of TILs gatedon live singlets from3patientswith ovarian cancer. Mapping byPD-1 and ICOS (top) or CD25 andCD127 (bottom), in addition toCD3, CD4, andCD8but not FOXP3 (both). Colors indicate expression level of FOXP3. I, In vitro suppression by intratumoral PD-1intICOShi Treg cells. Pooled results from 3 patients areshown. J, TSDRmethylation of the indicated intratumoral CD4þ T-cell subsets from 3patients. Each box represents an individual CpGmotif; each row represents onepatient. Percentage of DNA methylation at each motif is color-coded according to the scale. K, Hierarchical clustering of genes differentially expressed byintratumoral CD4þ T-cell subsets as determined by RNA-seq. Colors show row-normalized Z-score of gene expression values (log2 TPM þ 1).

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Treg cell–specific demethylated region (TSDR) within theFOXP3 locus, whereas activated effector cells that transientlyupregulate FOXP3 remain methylated (20). IntratumoralPD-1intICOShi cells displayed a demethylated TSDR (approxi-mately 50% methylation due to X-chromosome hypermethyla-tion in female patients), thus confirming that this subset con-stitutes bona fide Treg cells with epigenetically imprinted stableFOXP3 expression, whereas the TSDR of PD-1hiICOSint andPD-1-ICOS� cells was methylated (Fig. 1J). Transcriptomicprofiling by RNA-seq revealed that PD-1intICOShi cells had adistinct gene expression profile (Fig. 1K; SupplementaryTable S2). We also analyzed Treg cells isolated from PBMC byRNA-seq and found that although the overall gene expressionpattern was quite distinct from all intratumoral subsets, expres-sion of canonical Treg cell signature genes was shared betweencirculating and intratumoral Treg cells (Supplementary Fig. S2).Taken together, these results showed that tumor-infiltratingCD4þPD-1intICOShi T cells were bona fide Treg cells with potentsuppressive activity and a unique transcriptomic program.

We next addressed the question whether expression of addi-tional TCR-induced receptors was different in intratumoral Tregcells comparedwith peripheral blood. The proportion of FOXP3þ

Treg cells within tumor-infiltrating T cells and FOXP3 expressionon a per cell basis wasmarkedly increased compared with periph-eral blood (Fig. 2A–C). Moreover, expression of 4-1BB, CTLA-4,and OX40 was higher on intratumoral Treg cells compared withTreg cells fromperipheral blood (Fig. 2D–F). TIM-3was expressedat elevated levels on intratumoral Treg cells in some patients butshowed high interpatient variability (39.5% � 21.9% for intra-tumoral Treg cells and 21.8% � 12.8% for peripheral bloodTreg cells, Fig. 2G). The majority of circulating Treg cells wereTIGITþ, but the proportion of TIGIT-expressing cells and TIGITexpression levels were further enhanced in intratumoral Tregcells (Fig. 2H). In line with these phenotypic changes, the geneexpression pattern of intratumoral Treg cells was distinct fromcirculating Treg cells (Fig. 2I). Taken together, these resultsdemonstrate that Treg cells from tumors have substantial changesto their transcriptomic program and an altered immunoreceptor

Figure 2.

Intratumoral Treg cells display a distinct expression pattern of costimulatory and inhibitory receptors and a distinct transcriptional signature. A–C, Theexpression of intracellular FOXP3 in PBMCs and TILs frompatientswith ovarian cancerwas analyzed by flow cytometry. Representative dot plots on gatedCD3þ cells(A), summary data depicting the percentage of FOXP3þ cells within the CD3þ gate (B), and levels of FOXP3 expression (C) are shown (n ¼ 10). D–H,The expression of cell surface 4-1BB, TIM-3, OX40, and TIGIT as well as intracellular CTLA-4 by Treg cells in ovarian cancer specimens and peripheral blood wasanalyzed by flow cytometry. Representative contour plots on CD3þCD4þ gated TILs (left) and the percentage of intratumoral and circulating FOXP3þ cellsexpressing the indicated markers (right) are shown (n¼ 10). Expression levels of TIGIT on gated FOXP3þ cells are shown in H, right. Error bars, SD. �� , P < 0.0001,Mann–Whitney U test. I, Hierarchical clustering of differentially expressed genes in intratumoral Treg cells from patients with ovarian cancer and circulatingTreg cells from healthy donors as determined by RNA-seq. Colors show row-normalized Z-score of gene expression values (log2 TPM þ 1).






expression pattern. However, receptor upregulation occurred in aselective manner, as the percentage expressing CD27, CD28, andDNAM-1 was either reduced or not drastically changed (Supple-mentary Fig. S3).

We next examined the prevalence of na€�ve and effector Tregcells and the surface phenotype of these Treg cell subpopulationswithin ovarian tumors. Miyara and colleagues have categorizedhuman FOXP3-expressing regulatory and nonregulatory cellsbased on the expression levels of FOXP3 and CD45RA (21). Wehave found that CD45RA�FOXP3hi effector Treg cells were themost prevalent subset within ovarian tumors, whereasCD45RAþFOXP3lo na€�ve Treg cells were essentially absent(Fig. 3A and B). Within tumors both CD45RA�FOXP3hi effectorTreg cells and CD45RA�FOXP3lo effector T cells expressed 4-1BB,ICOS, OX40, and CTLA-4, but expression of these markers on theformer subset was higher (Fig. 3C). Strikingly, intratumoralFOXP3hi cells expressed intermediate levels of PD-1, whereas theFOXP3lo subset was either PD-1hi or PD-1�, akin to FOXP3� cells(Fig. 3D and E), suggesting that bona fide FOXP3hi effectorTreg cells display a characteristic cell surface phenotype withinovarian tumors that is distinct from FOXP3lo-activated effectorcells. Therefore, the gating strategy using FOXP3 and CD45RAshows that FOXP3hiCD45RA� Treg cells in the tumor expresshigher levels of 41BB, ICOS, OX40, CTLA4, and intermediatelevels of PD-1 compared with the FOXP3lo-activated effectorpopulation.

Mass cytometry analysis reveals a partially overlappingphenotype of Treg cells and conventional T-cell subsets

The distinct expression pattern of costimulatory and inhibitoryreceptors by tumor-infiltrating Treg cells prompted us to furtherexplore their phenotype. To this end, we performed in-depthanalyses of CD4þ T-cell subsets in ovarian cancer specimens by35-parameter mass cytometry (Supplementary Fig. S4A; Supple-mentary Table S3). Similar to our flow cytometry analyses, wecould detect PD-1hiICOSint, PD-1intICOShi, and PD-1�ICOS�

subsets, although overall PD-1 expression was lower by masscytometry (Supplementary Fig. S4B). PD-1�ICOS� resting T cells(Trest) did not express TIM-3 or TIGIT, whereas PD-1hiICOSint–activated T cells (Tact) and PD-1intICOShi Treg cells were TIM-3loTIGIThi (Fig. 4A). Trest cells also expressed the lowest levels ofCD27, CD28, and Ki-67 among the three subpopulations. Incontrast, Tact and Treg cells expressed higher levels of thesemolecules and expressionofCD27andCD28was further elevatedon Treg comparedwith Tact cells (Fig. 4A). In addition, 4-1BBwasdetectable only on Treg cells but none of the other subsets. Incontrast, expression levels of the chemokine receptors CXCR3 andCCR5, the costimulatory molecule DNAM-1, the checkpointmolecule PD-L1, and the transcription factor T-bet were similarin all subsets. Moreover, all subsets lacked the expression of theinhibitory receptor BTLA and CD160, but were uniformlyCD45RA�CD45ROþCD69þ (Fig. 4A and data not shown). Thesefindings demonstrate that different subsets of T cells foundwithin the tumor microenvironment can be distinguished by theexpression of markers associated with T-cell activation orexhaustion.

To further explore the activated Treg cell phenotype, we ana-lyzed our mass cytometry data using dimensionality reductionalgorithms. In line with an activation-dependent tuning of thetumor-infiltrating CD4þ T-cell phenotype, ViSNE analysisrevealed that Tact and Treg cells exhibited more similarities ona global level than either subset did with Trest cells (Fig. 4B).Notably, prominent populations of actively proliferating Ki-67þ

cells fell within the regions of the maps that coexpressedTact- and Treg cell-associated markers (Fig. 4B); hence, activelyproliferating CD4þ T-cell subsets were characterized by thecoexpression of several costimulatory and inhibitory receptorsregardless of suppressive function. In summary, these findingsfurther substantiated the high prevalence of T cells with anactivated phenotype within the tumor, and the phenotype ofintratumoral Treg cells in particular was indicative of a highlyactivated state.

Figure 3.

Expression of costimulatory and inhibitory receptors ismost pronounced in FOXP3hi effector Treg cells. A,Categorization of CD4þ T cells into CD45RAþFOXP3lo

na€�ve Treg (nTreg) cells, CD45RA�FOXP3hi effectorTreg (eTreg) cells, and CD45RA�FOXP3lo activatedeffector T (aTeff) cells. Plots show CD3þCD4þ gate,and boxed numbers indicate percentage of cellswithin the corresponding gate. B, Summary of resultsshown in A from 10 patients. C, Percentage of cellswithin the subsets defined in A, which express theindicated receptors within TILs and PBMCs. Asummary of 10 patients is shown. D, GMFI of PD-1on the indicated subsets within TILs and PBMCs.E, Representative histogram showing theexpression of PD-1 on intratumoral FOXP3hi Treg cells,FOXP3lo activated effector cells, and FOXP3�

effector cells. Error bars, SD. �� , P < 0.01; �� , P < 0.001;�� , P < 0.0001, Mann–Whitney U test.

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Melanoma-infiltrating Treg cells exhibit distinct phenotypicdifferences from ovarian tumor–infiltrating Treg cells

We next examined whether this highly activated Treg cellphenotype could also be found in cancers that respond better toimmunotherapy and thus extendedourCD4þTreg cell analyses tomelanoma specimens. ViSNE analysis of mass cytometry datarevealed that melanoma-infiltrating Treg cells formed a moreheterogeneous population (Fig. 5A and B). This heterogeneitybecame evident from the relatively diffuse distribution pattern ofmelanoma-infiltrating Treg cells over the entire CD4þ T-cellregion on the ViSNE map, whereas ovarian cancer Treg cellsformedmore distinct clusters. Among the dominant markers thatdefined this Treg cell cluster in both cancerswere ICOS, TIGIT, andKI-67 (Fig. 5A). To further elucidate the Treg cell heterogeneity, weperformed SPADE analysis on the samemass cytometry dataset of

ovarian cancer and melanoma specimens and discovered twosubpopulations of intratumoral FOXP3þ Treg cells. One of thesesubpopulations expressed higher levels of FOXP3, 4-1BB, ICOS,TIGIT, PD-1, CD25, TIM-3, and CD28 and is therefore referred toas Treg-hi, whereas the other subpopulation with lower expres-sion of all of these markers is referred to as Treg-lo (Fig. 5C;Supplementary Fig. S5A and S5B and data not shown). In linewith this observation, expression of select markers including4-1BB, PD-1, and ICOSwas increased in ovarian cancer Treg cells,although the frequency of tumor infiltration by Treg cells amongtotal T cells was not different from melanoma (Fig. 5D–G;Supplementary Fig. S5C and S5D). Accordingly, the Treg-hisubpopulation comprised a higher percentage of T cells and theratio of Treg-hi to Treg-lo was significantly increased withinovarian tumors compared with melanoma (Fig. 5H and I). Taken

Figure 4.

Mass cytometry analysis reveals partially overlapping expression of immunemarkers by intratumoral regulatory and nonregulatory T cells.A, Thirty- five–parametermass cytometry was performed on ovarian cancer specimens. Heatmaps show expression of the indicated markers by CD4þ T-cell subsets from 4 patients.Similar results were obtained in two independent experiments (n ¼ 8). B, ViSNE analysis of mass cytometry data from A. Data from 3 patients were used torun the algorithm on CD3þCD4þ cells; ViSNE maps are shown for one representative patient. Expression of markers shown above each individual map arehighlighted according to the color scheme. Gates indicate Treg, Tact, and Trest cell areas of the maps as depicted in the top left map.






together, thesefindings showed that althoughovarian tumors andmelanomas are infiltrated by similar proportions of FOXP3þ Tregcells, these infiltrates were qualitatively different. Importantly,these differences did not reflect global phenotypic differences ofthe T-cell infiltrate as PD-1 expression by CD8þ T cells was notdifferent (Supplementary Fig. S6).

The Treg-hi phenotype defines highly responsive Treg cellswith superior suppressive capacity

We next sought to characterize the implications of the Treg-hiandTreg-lo phenotype on the immune regulatory function of Tregcells. To this end, we selected two receptors that showed markeddifferential expression between the two Treg cell subpopulations.

Figure 5.

Mass cytometry analysis reveals two subpopulations of intratumoral Treg cells. A and B, ViSNE analysis of mass cytometry data on ovarian cancer andmelanoma specimens. ViSNE was run on CD3þ cells either excluding (A) or including FOXP3 (B) from mapping parameters as described in theMaterials and Methods section. Expressions of markers shown above each individual map are highlighted according to the color scheme. Maps are gatedon CD3þ cells (left panels showing CD4 expression) or FOXP3þ cells (remaining panels). Clustering was performed on data from 3 patients, and ViSNEmaps from one patient are shown. C, Mass cytometry data from ovarian cancer and melanoma specimens were pregated on CD3þ cells and analyzedby SPADE. Expression of ICOS is highlighted according to the rainbow scale in each individual tree. Data from 4 patients were used to generate SPADE trees;data from one representative patient per group are shown. Similar results were obtained in two independent experiments (n ¼ 4). D, Percentage ofintratumoral FOXP3þ Treg cells among CD3þ cells in ovarian cancer and melanoma. E, GMFI of FOXP3 expression by Treg cells (n ¼ 10). F, Percentageof 4-1BBþ cells among Treg cells in ovarian cancer and melanoma. G, GMFI of the indicated markers on gated Treg cells in ovarian cancer andmelanoma was analyzed by flow cytometry (n ¼ 10). H, The relative abundance of cells within the Treg-hi and Treg-lo clusters as defined by SPADEanalysis in ovarian cancer and melanoma is depicted as frequency within CD3þ cells. I, The ratio of cells within the Treg-hi and Treg-lo clusters inovarian cancer and melanoma (n ¼ 4). Error bars, SD. �� , P < 0.0001; �� , P < 0.01; � , P < 0.05; Mann–Whitney U test.

Toker et al.





First, we correlated the responsiveness of Treg cells to TCR stim-ulation with their PD-1 expression levels. We purified Treg cellsfrom ovarian tumors and cultured them for one day eitherwithout TCR stimulation or with high or low concentration ofimmobilized anti-CD3/anti-CD28 antibodies. A minor fractionof unstimulated Treg cells expressed 4-1BB after one day, whereas4-1BB expression increased after stimulation in a concentration-dependent manner along with downregulation of the TCR com-plex (Fig. 6A). Importantly, Treg cells expressing relatively highlevels of PD-1 were more readily responsive to a low dose of TCRstimulation, such that upregulation of 4-1BB was not substan-tially more pronounced at the high-dose TCR stimulation(Fig. 6B). These results suggested that PD-1 expression levelscould be used as a correlate of activation state in tumor-infiltratingTreg cells.

Wenext investigated the significance of ex vivo 4-1BB expressionon the functional capacity of Treg cells. We purified 4-1BBþ and4-1BB� Treg cells from ovarian cancer specimens and assessed

their functional capacity in an in vitro suppression assay. As shownin Fig. 6C, 4-1BBþ Treg cells had superior suppressive capacitycompared with patient-matched 4-1BB� Treg cells. In agreementwith these findings, bulk Treg cells purified from ovarian tumorshad enhanced suppressive capacity when compared with mela-noma-infiltrating Treg cells (Fig. 6D). These results showed thatthe immunologic receptor expression pattern including increased4-1BB, PD-1, and ICOS expression defines highly activated andsuppressive Treg cells that infiltrate ovarian tumors.

DiscussionTumor-infiltrating Treg cells have an activated phenotype

Peripheral blood Treg cells are a heterogeneous population thatcanbe subdivided into several partially overlapping subsets by thedifferential expression CD45RA, CCR4, CD39, CD25, and others(19, 21). Circulating Treg cells also express some costimulatoryand inhibitory receptors such as CD27 and CD28, but manyothers like ICOS, OX40, 4-1BB, TIM3, and PD-1 are absent orexpressed at low levels ex vivo (19, 22). In contrast, tumor-infiltrating Treg cells express CD45RO and increased levels of theeffector molecules CD39 and CD73, indicating previous antigenexperience and an elevated activation state (23). However, otheraspects of the intratumoral Treg cell phenotype with potentialclinical relevance, such as expression of costimulatory and inhib-itory receptors, have not been comprehensively studied.

We report that the array of such immunologic receptorsexpressed by intratumoral Treg cells is strikingly different fromcirculating Treg cells, suggesting that substantial phenotypicalchanges occur concomitantly with tumor infiltration. Our find-ings are supported by two recent studies that showed that Tregcells isolated from breast cancer, non–small cell lung cancer, andcolorectal cancer have distinct transcriptomic signatures com-pared with peripheral blood Treg cells (24, 25). Genes upregu-lated in the tumor included costimulatory and cytokine receptorsas well as trafficking and effector molecules, suggesting thattumor-infiltrating Treg cells share common features across can-cers. Our transcriptomic and mass cytometry analyses show thatmany of these features are in fact shared between Treg cells andCD4þ effector T cells, suggesting that a large proportion of tumor-infiltrating CD4þ T cells are activated and potentially involved inantitumor immunity. These observations are in line with a recentstudy that showed that tissue-specific transcriptomic signaturesfrom CD4þ T-cell subsets are dominant over lineage-specific (i.e.,regulatory vs. effector) signatures in liver cancer (26). However, inspite of the common tumor-associated gene expression profile,key cell subset-specific features such as suppressive functionremain intact and may even be enhanced within the tumormicroenvironment. Indeed, we show that elevated expression ofPD-1 and 4-1BBby intratumoral Treg cells correlatedwith respon-siveness to TCR stimulation and suppressive capacity. In agree-ment with our findings, previous reports have shown that theexpression of PD-1 and TIM-3 correlated with increased IL10production by Treg cells (27). In addition, TIGITþTIM-3þ Tregcells exhibit enhanced immunosuppression in a murine tumormodel (28), and the presence of a TIM-3–expressing subset oftumor-infiltrating Treg cells has been associated with progressionin non–small cell lung cancer (22). Furthermore, infiltration ofprimary cutaneous squamous cell carcinomasbyOX40þTreg cellswas associated with a higher incidence of metastasis (29),suggesting that a highly activated intratumoral Treg cell

Figure 6.

Treg cells expressing PD-1 and 4-1BB are highly responsive to stimulationand have superior suppressive capacity. A, CD4þPD-1intICOShi Treg cellswere purified from ovarian tumors and stimulated with a lowconcentration or high concentration of immobilized anti-CD3/anti-CD28antibodies or left unstimulated. Expression of CD3 and 4-1BB was evaluatedafter 24 hours by flow cytometry. B, Linear regression analysis (n ¼ 10)of Treg cells stimulated as in A. The difference in 4-1BB expressionbetween high- and low-dose CD3/CD28 stimulation is shown in relationto ex vivo PD-1 expression levels. C, The suppressive capacity ofCD4þPD-1intICOShi4-1BBþ and 4-1BB� Treg cells from ovarian tumors wasassessed in vitro (n ¼ 4). �� , P < 0.01; � , P < 0.05, unpaired t test. D, Thesuppressive capacity of Treg cells purified from ovarian cancer andmelanoma tissues was assessed in vitro. The ratio of responder T cells toTreg cells was 1:2 (n ¼ 7–8). �, P < 0.05, Mann–Whitney U test.






phenotype is defined by both activating and inhibitory receptorsthat are induced by TCR stimulation. These findings are allconsistent with the interpretation that TCR-triggered Treg cellsas defined by expressing one or more of these markers includingPD-1, 41BB, TIM3, TIGIT, or OX-40 have increased immunosup-pressive function.

Treg cells exhibit distinct features in ovarian cancer andmelanoma

Although many studies investigating intratumoral Treg cellphenotype were focused on the expression of individual receptorsin different cancers, a high degree of overlap in transcriptomicsignatures from tumor-infiltrating Treg cells across cancers sug-gests that some factors that shape the Treg cell phenotype areshared between malignancies (24–26). However, comparativetranscriptomic analyses also showed that Treg cells in hepatocel-lular carcinoma, non–small cell lung cancer, and breast cancerexhibit partially distinct gene expression programs (26), suggest-ing that substantial heterogeneity among Treg cells infiltratingdifferent cancers may exist beyond a common tumor-associatedcore gene expression signature.Wenowextendon these studies bydirectly showing that a variety of costimulatory and coinhibitorymolecules are differentially expressed in ovarian cancer andmelanoma.

Treg cells infiltrating ovarian cancers displayed a Treg-hiphenotype that was characterized by higher expression of FOXP3,PD-1, 4-1BB, ICOS, CD25, and other markers in comparisonwith the Treg-lo phenotype, which was enriched in melanoma.Individual features of the Treg-lo phenotype have previouslybeen associated with increased plasticity and effector T-cell func-tion. For instance, FoxP3þCD25� murine Treg cells are proneto conversion to effector T cells in response to inflammatorycues (30). Indeed, a substantial fraction of melanoma antigen-specific FOXP3þ cells can secrete Th1-type cytokines followingimmunotherapy in patients with melanoma (31). Moreover,CD45ROþFOXP3lo effector T cells with increased capacity forIFNg and TNFa production accumulate in the peripheral blood ofpatients with melanoma (32). Infiltration by CD4þFOXP3lo cellsis associated with improved antitumor immunity and survival incolorectal cancer (33). Therefore, a higher prevalence of the Treg-lo phenotype and reduced in vitro suppressive capacity suggeststhat FOXP3þ cells in melanoma are heterogeneous, and includenonregulatory cells and plastic Treg cells with effector T-cellpotential. Importantly, melanoma-infiltrating Treg cells dis-played a T-betþCXCR3þ tumor migratory phenotype and con-tained a Ki-67þ subset, suggesting that they were specificallyrecruited to the tumor and recognizing tumor antigen in situ.Such tumor-specific cells with reduced suppressive function andproinflammatory potential could play a beneficial role in animmunotherapeutic setting and contribute to the comparativelyhigh response rates in melanoma (2, 34).

The potential of Treg cell–targeted immunotherapiesExtensive immunosuppression owing to the Treg-hi

phenotype in ovarian cancer described here as well as TAM(7) or innate lymphoid cells (35) could explain the highresistance of ovarian cancer to immunotherapy despite pro-nounced CD8þ T-cell infiltration (2, 36). Targeting Treg cell–suppressive function has gained much interest as an avenueto promote tumor immunotherapy, but challenges remainsuch as the need to selectively manipulate tumor-infiltrating

Treg cells. It is desirable that Treg cell depleting or modulatingimmunotherapies spare circulating Treg cells with broadself-antigen specificity, thereby avoiding a systemic breachin self-tolerance and the occurrence of autoimmunity (5).Depletion of Treg cells by targeting the chemokine receptorCCR4 can induce melanoma antigen-specific immuneresponses in vitro (37), but the mechanism of action as wellas the specificity of this approach to intratumoral Treg cellsremain incompletely understood. Several studies in animalmodels have shown that intratumoral Treg cells can be selec-tively depleted by antibodies that target CTLA-4, GITR, andOX40, owing to the elevated expression of these markers bytumor-infiltrating Treg cells (38). It has been suggested that themechanism of action of ipilimumab in patients with melano-ma may involve depletion of CTLA-4 expressing Treg cellsthrough antibody-dependent cell-mediated cytotoxicity bynonclassical monocytes (39). Our observation that intratu-moral Treg cells express elevated levels of CTLA-4 provides apotential mechanism for their selective depletion after ipili-mumab treatment.

Our studies suggest that immunotherapies targeting othermolecules expressed by intratumoral Treg cells such as 4-1BB,OX40, and ICOS may act by depleting or modulating Tregcells. For instance, triggering ICOS on Treg cells throughICOSL-expressing tumor cells or plasmacytoid dendritic cellsboosts suppressive capacity (40, 41); hence, blockade of thispathway could attenuate Treg cell function. In contrast, stud-ies with murine Treg cells have shown that signaling throughthe inhibitory receptors TIGIT and PD-1 can attenuate theirsuppressive function (28, 42). Therefore, blockade of inhib-itory receptors may have the unwanted side effect of increasedTreg cell activity. These considerations are particularly impor-tant because many mAbs that target checkpoint receptors thatare coexpressed by effector T cells and Treg cells are currentlyapproved or undergoing clinical evaluation for the treatmentof cancer (3, 4). Such immunomodulating agents often actthrough multiple mechanisms, and in most cases therelative contribution of each mechanism to the therapeuticeffect is unknown (43). Development of immune therapeuticapproaches will therefore require careful consideration of theeffect of the manipulating agent on both effector T cells andTreg cells.

Conventional chemotherapies can have profound effects onthe immune system and alter the composition of immune cellswithin the tumor microenvironment (44). Chemotherapy fre-quently results in immunogenic cancer cell death that is accom-panied by release of antigens and antigen-presenting cell mat-uration signals (44). Moreover, neoadjuvant chemotherapyinduces NF-kB–driven upregulation of MHC class I and PD-L1by cancer cells and increased T-cell infiltration of ovariantumors (45–47); hence, the idea of reinforcing chemothera-py-induced tumor-specific immune responses through check-point blockade has gained much interest (48). Some conven-tional chemotherapeutic agents such as cyclophosphamide anddocetaxel can also selectively reduce Treg cells (49, 50), andreduction of intratumoral Treg cells after neoadjuvant chemo-therapy is associated with improved responses (47), suggestingthat a window of opportunity may exist where CD8-targetedimmunotherapies could exploit a temporary reduction inimmunosuppressive Treg cells following chemotherapy. Alter-natively, combining chemotherapies that deplete MDCSs (44)

Toker et al.





with Treg cell–targeted immunotherapies may cause a moreprofound ablation in immunosuppressive cell populations andcreate a tumor microenvironment that is highly favorable forantitumor immunity.

The search for rational combination (immuno)-therapiesfrom an ever-growing pool of available options needs to takeinto account several factors including the expression pattern ofthe target and its potential mechanism of action. We now reportthe expression of various costimulatory and coinhibitory recep-tors by Treg cells is highly relevant to therapeutic approachescurrently in use or under development, adding another facet tothe potential mechanism of action of these agents. The possi-bility of Treg cell modulation is therefore an additional factorthat needs to be taken into consideration in the design ofcombination therapies.

Disclosure of Potential Conflicts of InterestT.J. Pugh reports receiving other commercial research support from

Boehringer Ingelheim and speakers bureau honoraria from Merck, and isa consultant/advisory board member for Chrysalis Biomedical Advisors andDynaCare/Impact Genetics. M.Q. Bernardini is a consultant/advisory boardmember for Astra Zeneca. No potential conflicts of interest were disclosed bythe other authors.

Authors' ContributionsConception and design: A. Toker, L.T. Nguyen, P.S. OhashiDevelopment of methodology: A. Toker, C.J. Guidos, T.J. Pugh

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P.A. Shaw, D. Ghazarian, A. Al-Habeeb, A. Easson,W.L. Leong, M. Reedijk, C.J. Guidos, T.J. Pugh, M.Q. BernardiniAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, com-putational analysis): A. Toker, S.Y.C. Yang, B.A. Clarke, C.J. Guidos, T.J. PughWriting, review, and/or revision of the manuscript: A. Toker, S.Y.C. Yang,S.R. Katz, A. Al-Habeeb, A. Easson, D.R. McCready, M. Reedijk, T.J. Pugh,P.S. OhashiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.R. Katz, M.Q. BernardiniStudy supervision: T.J. Pugh, P.S. OhashiOther (conducted experiments): S.C. Stone

AcknowledgmentsThis work was supported by a CIHR Foundation award to P.S. Ohashi and a

TRI award sponsored by OICR to P.S. Ohashi and the Garron Family CancerCentre to C.J. Guidos. We thank the Flow andMass Cytometry Facility (FMCF),The Hospital for Sick Children, Toronto, Canada, for assistance with cell sortingand mass cytometry data acquisition. The authors thank Diana Gray forproviding patient data. They also thank the patients and their families foragreeing to provide the tissue for research.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 15, 2018; revised June 18, 2018; accepted July 26, 2018;published first July 31, 2018.

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Toker et al.




Published OnlineFirst July 31, 2018.Clin Cancer Res Aras Toker, Linh T. Nguyen, Simone C. Stone, et al. Highly Activated Phenotype Distinct from that in MelanomaRegulatory T Cells in Ovarian Cancer Are Characterized by a

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